Soluble pharmaceutical compositions for parenteral administration comprising a GLP-1 peptide and an insulin peptide of short time action for treatment of diabetes and bulimia

ABSTRACT

Pharmaceutical composition for parenteral administration comprising a meal related insulin peptide and an insulinotropic peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application serialno. PCT/DK2004/000788 filed Nov. 12, 2004 and claims priority fromDanish Application serial no. PA 2003 01689 filed Nov. 13, 2003 and ofU.S. provisional application Ser. No. 60/519,590 filed on Nov. 13, 2003.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticalcompositions. More specifically the invention pertains to pharmaceuticalcompositions comprising two different pharmaceutically active peptides.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder in which the ability toutilize glucose is partly or completely lost. About 5% of all peoplesuffer from diabetes and the disorder approaches epidemic proportions.Since the introduction of insulin in the 1920's, continuous efforts havebeen made to improve the treatment of diabetes mellitus. Since peoplesuffering from diabetes are subject to chronic treatment over severaldecades, there is a major need for safe, convenient and life qualityimproving insulin formulations.

In the treatment of diabetes mellitus, many varieties of insulinformulations have been suggested and used, such as regular insulin,isophane insulin (designated NPH), insulin zinc suspensions (such asSemilente®, Lente®, and Ultralente®), and biphasic isophane insulin.Some of the commercial available insulin formulations are characterizedby a fast onset of action and other formulations have a relatively slowonset but show a more or less prolonged action. Fast-acting insulinformulations are usually solutions of insulin, while retarded actinginsulin formulations can be suspensions containing insulin incrystalline and/or amorphous form precipitated by addition of zinc saltsalone or by addition of protamine or by a combination of both.

Normally, insulin formulations are administered by subcutaneousinjection. What is important for the patient is the action profile ofthe insulin formulation which is the action of insulin on the glucosemetabolism as a function of the time from the injection. In thisprofile, inter alia, the time for the onset, the maximum value, and thetotal duration of action are important. A variety of insulinformulations with different action profiles are desired and requested bythe patients.

Human insulin consists of two polypeptide chains, the so-called A and Bchains which contain 21 and 30 amino acid residues, respectively. The Aand B chains are interconnected by two cystine disulphide bridges.Insulin from most other species has a similar construction, but may notcontain the same amino acid residues at the same positions. Within thelast decade a number of human insulin analogues have been developed.They are designed for particular profiles of action, i.e. fast acting orprolonged action.

Another peptide expected to become very important in the treatment ofdiabetes is glucagon-like peptide-1 (GLP-1). Human GLP-1 is a 37 aminoacid residue peptide originating from preproglucagon which issynthesized i.a. in the L-cells in the distal ileum, in the pancreas andin the brain. GLP-1 is an important gut hormone with regulatory functionin glucose metabolism and gastrointestinal secretion and metabolism.GLP-1 stimulates insulin secretion in a glucose-dependant manner,stimulates insulin biosynthesis, promotes beta cell rescue, decreasesglucagon secretion, gastric emptying and food intake. A simple system isused to describe fragments and analogues of this peptide. Thus, forexample, Gly⁸-GLP-1(7-37) designates an analogue of GLP-1(7-37) formallyderived from GLP-1(7-37) by substituting the naturally occurring aminoacid residue in position 8 (Ala) by Gly. Similarly,Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-37) whereinthe ε-amino group of the Lys residue in position 34 has beentetradecanoylated. PCT publications WO 98/08871 and WO 99/43706 disclosestable derivatives of GLP-1 analogues, which have a lipophilicsubstituent. These stable derivatives of GLP-1 analogues have aprotracted profile of action compared to the corresponding GLP-1analogues.

As the type 2 diabetes population is rapidly increasing in the world,there is a much larger need for simpler administration of more effectivedrugs. The combined effects of GLP-1 are expected to give very effectiveand safe lowering of blood glucose. However, some patients may benefitfrom an extra small dose of insulin with the main meals. A combinationformulation comprising an insulin peptide and a GLP-1 peptide, may witha fixed ratio of the two pharmaceuticals, be a very efficacioustreatment as well as one requiring less injections when administered tothe same patient. Because only a low dose of insulin is given with themeal and the GLP-1 counterpart of the formulation controls glucose forthe rest of the day and night, and since GLP-1 does not lead tohypoglycaemia it may also be a very safe treatment.

Thus, there is a big need for stable pharmaceutical compositionscomprising meal-related insulin and a GLP-1 peptide in one combinedformulation.

BREIF DESCRIPTION OF THE DRAWINGS

FIG. 1. Average disappearance curves showing the disappearance ofradiolabeled insulin aspart.

FIG. 2. Plasma levels of liraglutide after subcutaneous injections ofthe mixtures (logarithmic scale).

FIG. 3. Plasma levels of liraglutide after subcutaneous injections ofthe mixtures (linear scale).

FIG. 4. Turbidimetric measurements of two pharmaceutical compositionscontaining aspart and liraglutide. Pharmaceutical composition withoutpoloxamer 188 (x) and with poloxamer 188 (⋄) (500 ppm).

FIG. 5. Turbidimetric measurements of two pharmaceutical compositionscontaining aspart and liraglutide. Pharmaceutical composition withoutpoloxamer 188 (x) and with poloxamer 188 (⋄) (50 ppm).

FIG. 6. All samples contain: 0.6 mM insulin aspart, 0.3 mM Zn(Ac)₂, 1.2mM Liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 10 mM NaCl, pH7.7. Poloxamer 188 is added to two of the samples.

FIG. 7. Both samples contain 0.6 mM insulin aspart, 0.3 mM Zn(Ac)₂, 1.2mM Liraglutide, 40 mM phenol, 14 mg/ml propylene glycol, 10 mM NaCl, pH7.7. Polysorbate 20 is added to one sample.

FIG. 8. All samples contain 0.6 mM insulin aspart, 0.3 mM Zn(Ac)₂, 1.2mM Liraglutide, 8 mM sodium phosphate pH 7.7, 40 mM phenol, 14 mg/mlpropylene glycol. Poloxamer 188 is added to two samples.

FIG. 9. 0.6 mM insulin aspart, 0.3 mM Zn(Ac)₂, 1.2 mM Liraglutide, 8 mMsodium phosphosfate, 40 mM phenol, 14 mg/ml propylene glycol, pH 7.7.Polysorbate 20 is added to two of the samples.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a solublepharmaceutical composition for parenteral administration, whichcomprises an insulinotropic peptide, a meal related insulin peptide, apharmaceutically acceptable preservative and optionally an isotonicityagent. In one embodiment of the invention, pH of said pharmaceuticalcomposition or a reconstituted solution of said pharmaceuticalcomposition is from about pH 7.0 to about pH 9.0. In another embodimentof the invention, pH of said pharmaceutical composition or areconstituted solution of said pharmaceutical composition is from aboutpH 7.0 to about pH 8.0.

Another object of the invention is to provide a method for the treatmentof hyperglycemia comprising parenteral administration of an effectiveamount of a soluble pharmaceutical composition comprising aninsulinotropic peptide, a meal-related insulin peptide, apharmaceutically acceptable preservative and optionally an isotonicityagent.

Definitions

The following is a detailed definition of the terms used in thespecification.

The term “effective amount” as used herein means a dosage which issufficient in order for the treatment of the patient to be effectivecompared with no treatment.

The term “medicament” as used herein means a pharmaceutical compositionsuitable for administration of the pharmaceutically active compounds toa patient.

The term “pharmaceutical composition” as used herein means a productcomprising an active compound or a salt thereof together withpharmaceutical excipients such as buffer, preservative and tonicitymodifier, said pharmaceutical composition being useful for treating,preventing or reducing the severity of a disease or disorder byadministration of said pharmaceutical composition to a person. Thus apharmaceutical composition is also known in the art as a pharmaceuticalformulation.

The term “soluble pharmaceutical composition” as used herein means aninsulinotropic peptide which is substantially soluble, and ameal-related insulin peptide which is substantially soluble in thecombined composition. Thus, a predisssolved soluble pharmaceuticalcomposition will be substantially soluble, and a soluble pharmaceuticalcomposition which is to be reconstituted will be substantially solubleonce it has been dissolved in the prescribed reconstitution liquid. Itis to be understood that pH of a pharmaceutical composition which is tobe reconstituted is the pH value which is measured on the reconstitutedcomposition produced by reconstitution in the prescribed reconstitutionliquid at room temperature.

The term “pharmaceutically acceptable” as used herein means suited fornormal pharmaceutical applications, i.e. giving rise to no adverseevents in patients etc.

The term “buffer” as used herein refers to a chemical compound in apharmaceutical composition that reduces the tendency of pH of thecomposition to change over time as would otherwise occur due to chemicalreactions. Buffers include chemicals such as sodium phosphate, TRIS,glycine and sodium citrate.

The term “preservative” as used herein refers to a chemical compoundwhich is added to a pharmaceutical composition to prevent or delaymicrobial activity (growth and metabolism). Examples of pharmaceuticallyacceptable preservatives are phenol, m-cresol and a mixture of phenoland m-cresol.

The term “isotonicity agent” as used refers to a chemical compound in apharmaceutical composition that serves to modify the osmotic pressure ofthe pharmaceutical composition so that the osmotic pressure becomescloser to that of human plasma. Isotonicity agents include NaCl,glycerol, mannitol etc.

The term “stabilizer” as used herein refers to chemicals added topeptide containing pharmaceutical compositions in order to stabilize thepeptide, i.e. to increase the shelf life and/or in-ude time of suchcompositions. Examples of stabilizers used in pharmaceuticalformulations are L-glycine, L-histidine, arginine, polyethylene glycol,and carboxymethylcellulose.

The term “treatment of a disease” as used herein means the managementand care of a patient having developed the disease, condition ordisorder. The purpose of treatment is to combat the disease, conditionor disorder. Treatment includes the administration of the activecompounds to eliminate or control the disease, condition or disorder aswell as to alleviate the symptoms or complications associated with thedisease, condition or disorder.

The term “prevention of a disease” as used herein is defined as themanagement and care of an individual at risk of developing the diseaseprior to the clinical onset of the disease. The purpose of prevention isto combat the development of the disease, condition or disorder, andincludes the administration of the active compounds to prevent or delaythe onset of the symptoms or complications and to prevent or delay thedevelopment of related diseases, conditions or disorders.

The term “insulin peptide” as used herein means a peptide which iseither human insulin or a chemically modified human insulin, such as ananalog or a derivative thereof.

The term “human insulin” as used herein means the human hormone whosestructure and properties are well known. Human insulin has twopolypeptide chains that are connected by disulphide bridges betweencysteine residues, namely the A-chain and the B-chain. The A-chain is a21 amino acid peptide and the B-chain is a 30 amino acid peptide, thetwo chains being connected by three disulphide bridges: one between thecysteines in position 6 and 11 of the A-chain, the second between thecysteine in position 7 of the A-chain and the cysteine in position 7 ofthe B-chain, and the third between the cysteine in position 20 of theA-chain and the cysteine in position 19 of the B-chain.

The term “analogue” as used herein referring to a peptide means amodified peptide wherein one or more amino acid residues of the peptidehave been substituted by other amino acid residues and/or wherein one ormore amino acid residues have been deleted from the peptide and/orwherein one or more amino acid residues have been deleted from thepeptide and or wherein one or more amino acid residues have been addedto the peptide. Such addition or deletion of amino acid residues cantake place at the N-terminal of the peptide and/or at the C-terminal ofthe peptide.

The term “derivative” as used herein in relation to a parent peptidemeans a chemically modified parent protein or an analogue thereof,wherein at least one substituent is not present in the parent protein oran analogue thereof, i.e. a parent protein which has been covalentlymodified. Typical modifications are amides, carbohydrates, alkyl groups,acyl groups, esters, PEGylations and the like. Examples of derivativesof human insulin are threonine methyl ester^(B30) human insulin andN^(εB29)-tetradecanoyl des(B30) human insulin.

The term “meal-related insulin peptide” as used herein means an insulinpeptide which has a time-action of less than 8 hours in standard modelsof diabetes. Preferably, the meal-related human insulin has atime-action of less than about 5 hours. Preferably, the meal-relatedinsulin has a time-action in the range from 0 hours to about 4 hours.Preferably, the meal-related insulin has a time-action similar to thatobserved for commercial pharmaceutical compositions of Actrapid®,Novolog®, and Humalog®. The term about in relation to the time-action ofinsulins means + or −30 minutes.

The term “GLP-1 compound” as used herein means GLP-1(7-37) (SEQ ID NO.1), insulinotropic analogue thereof and insulinotropic derivativesthereof. Non-limiting examples of GLP-1 analogues are GLP-1(7-36) amide,Arg³⁴-GLP-1(7-37), Gly⁸-GLP-1(7-37), Val⁸-GLP-1(7-36)-amide andVal⁸Asp²²-GLP-1(7-37). Non-limiting examples of GLP-1 derivatives aredesamino-His⁷, Arg²⁶,Lys³⁴(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37), desamino-His⁷,Arg²⁶, Lys³⁴(N^(ε)-octanoyl)-GLP-1(7-37), Arg^(26,34), Lys³⁸(N^(ε)-(ω-carboxypentadecanoyl))-GLP-1(7-38), Arg^(26,34),Lys³⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1 (7-36) and Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1 (7-37).

The term “stable GLP-1 compound” as used herein means a chemicallymodified GLP-1(7-37), i.e. an analogue or a derivative which exhibits anin vivo plasma elimination half-life of at least 10 hours in man, asdetermined by the following method. The method for determination ofplasma elimination half-life of a peptide in man is: The compound isdissolved in an isotonic buffer, pH 7.4, PBS or any other suitablebuffer. The dose is injected peripherally, preferably in the abdominalor upper thigh. Blood samples for determination of active compound aretaken at frequent intervals, and for a sufficient duration to cover theterminal elimination part (e.g. Pre-dose, 1, 2, 3, 4, 5, 6, 7, 8, 10,12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3), 72 (day 4) and 84(day 4) hours post dose). Determination of the concentration of activecompound is performed as described in Wilken et al., Diabetologia43(51):A143, 2000. Derived pharmacokinetic parameteres are calculatedfrom the concentration-time data for each individual subject by use ofnon-compartmental methods, using the commercially available softwareWinNonlin Version 2.1 (Pharsight, Cary, N.C., USA). The terminalelimination rate constant is estimated by log-linear regression on theterminal log-linear part of the concentration-time curve, and used forcalculating the elimination half-life.

The term “dipeptidyl aminopeptidase IV protected GLP-1 compound” as usedherein means a GLP-1 compound which is more resistant to the plasmapeptidase dipeptidyl aminopeptidase IV (DPP-IV) than the native GLP-1agonist, GLP-1(7-37). Resistance of a GLP-1 compound towards degradationby dipeptidyl aminopeptidase IV is determined by the followingdegradation assay:

-   Aliquots of the GLP-1 compound (5 nmol) are incubated at 37° C. with    1 μL of purified dipeptidyl aminopeptidase IV corresponding to an    enzymatic activity of 5 mU for 10-180 minutes in 100 μL of 0.1 M    triethylamine-HCl buffer, pH 7.4. Enzymatic reactions are terminated    by the addition of 5 μL of 10% trifluoroacetic acid, and the peptide    degradation products are separated and quantified using HPLC    analysis. One method for performing this analysis is : The mixtures    are applied onto a Vydac C18 widepore (30 nm pores, 5 μm particles)    250×4.6 mm column and eluted at a flow rate of 1 ml/min with linear    stepwise gradients of acetonitrile in 0.1% trifluoroacetic acid (0%    acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48%    acetonitrile for 1 min) according to Siegel et al., Regul. Pept.    1999; 79:93-102 and Mentlein et al. Eur. J. Biochem. 1993;    214:829-35. Peptides and their degradation products may be monitored    by their absorbance at 220 nm (peptide bonds) or 280 nm (aromatic    amino acids), and are quantified by integration of their peak areas    related to those of standards. The rate of hydrolysis of a GLP-1    compound by dipeptidyl aminopeptidase IV is estimated at incubation    times which result in less than 10% of the GLP-1. compound being    hydrolysed.

The term “insulinotropic” as used herein referring to a peptide or acompound means the ability to stimulate secretion of insulin in responseto an increased plasma glucose level. Insulinotropic peptides andcompounds are agonists of the GLP-1 receptor. The insulinotropicproperty of a compound may be determined by in vitro or in vivo assaysknown in the art. The following in vitro assay may be used to determinethe insulinotropic nature of a compound such as a peptide. Preferablyinsulinotropic compounds exhibit an EC₅₀ value in below assay of lessthan 5 nM, even more preferably EC50 values less than 500 pM.

Baby hamster kidney (BHK) cells expressing the cloned human GLP-1receptor (BHK 467-12A) are grown in DMEM media with the addition of 100IU/mL penicillin, 100 μL/mL streptomycin, 10% foetal calf serum and 1mg/mL Geneticin G-418 (Life Technologies). Plasma membranes are preparedby homogenization in buffer (10 mM Tris-HCl, 30 mM NaCl and 1 mMdithiothreitol, pH 7.4, containing, in addition, 5 mg/mL leupeptin(Sigma), 5 mg/L pepstatin (Sigma), 100 mg/L bacitracin (Sigma), and 16mg/L aprotinin (Calbiochem-Novabiochem, La Jolla, Calif.)). Thehomogenate was centrifuged on top of a layer of 41% W7v sucrose. Thewhite band between the two layers was diluted in buffer and centrifuged.Plasma membranes were stored at −80° C. until used.

-   The functional receptor assay is carried out by measuring cAMP as a    response to stimulation by the insulinotropic peptide or    insulinotropic compound. Incubations are carried out in 96-well    microtiter plates in a total volume of 140 mL and with the following    final concentrations: 50 mM Tris-HCl, 1 mM EGTA, 1.5 mM MgSO₄, 1.7    mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1-methylxanthine (IBMX), 0.01%    w/v Tween-20, pH 7.4. Compounds are dissolved and diluted in buffer.    GTP is freshly prepared for each experiment: 2.5 μg of membrane is    added to each well and the mixture is incubated for 90 min at room    temperature in the dark with shaking. The reaction is stopped by the    addition of 25 mL 0.5 M HCl. Formed cAMP is measured by a    scintillation proximity assay (RPA 542, Amersham, UK). A    dose-response curves is plotted for the compound and the EC₅₀ value    is calculated using GraphPad Prism software.

The term “prodrug of an insulinotropic compound” as used herein means achemically modified compound which following administration to thepatient is converted to an insulinotropic compound. Such prodrugs aretypically amino acid extended versions or esters of an insulinotropiccompound.

The term “exendin-4 compound” as used herein is defined asexendin-4(1-39) (SEQ ID NO. 2), insulinotropic fragments thereof,insulinotropic analogs thereof and insulinotropic derivatives thereof.Insulinotropic fragments of exendin-4 are insulinotropic peptides forwhich the entire sequence can be found in the sequence of exendin-4 (SEQID NO. 2) and where at least one terminal amino acid has been deleted.Examples of insulinotropic fragments of exendin-4(1-39) areexendin-4(1-38) and exendin-4(1-31). The insulinotropic property of acompound may be determined by in vivo or in vitro assays well known inthe art. For instance, the compound may be administered to an animal andmonitoring the insulin concentration over time. Insulinotropic analogsof exendin-4(1-39) refer to the respective molecules wherein one or moreof the amino acids residues have been exchanged with other amino acidresidues and/or from which one or more amino acid residues have beendeleted and/or from which one or more amino acid residues have beenadded with the proviso that said analogue either is insulinotropic or isa prodrug of an insulinotropic compound . An example of aninsulinotropic analog of exendin-4(1-39) is Ser²Asp³-exendin-4(1-39)wherein the amino acid residues in position 2 and 3 have been replacedwith serine and aspartic acid, respectively (this particular analog alsobeing known in the art as exendin-3). Insulinotropic derivatives ofexendin-4(1-39) and analogs thereof are what the person skilled in theart considers to be derivatives of these peptides, i.e. having at leastone substituent which is not present in the parent peptide molecule withthe proviso that said derivative either is insulinotropic or is aprodrug of an insulinotropic compound. Examples of substituents areamides, carbohydrates, alkyl groups, esters and lipophilic substituents.An example of an insulinotropic derivatives of exendin-4(1-39) andanalogs thereof is Tyr³¹-exendin-4(1-31 )-amide.

The term “stable exendin-4 compound” as used herein means a chemicallymodified exendin-4(1-39), i.e. an analogue or a derivative whichexhibits an in vivo plasma elimination half-life of at least 10 hours inman, as determined by the method described under the definition of“stable GLP-1 compound”.

The term “dipeptidyl aminopeptidase IV protected exendin-4 compound” asused herein means an exendin-4 compound which is more resistant towardsthe plasma peptidase dipeptidyl aminopeptidase IV (DPP-IV) thanexendin-4 (SEQ ID NO. 2), as determined by the assay described under thedefinition of dipeptidyl aminopeptidase IV protected GLP-1 compound.

The term “isoelectric point” as used herein means the pH value where theoverall net charge of a macromolecule such as a peptide is zero. Inpeptides there may be several charged groups, and at the isoelectricpoint the sum of all these charges is zero. At a pH above theisoelectric point the overall net charge of the peptide will benegative, whereas at pH values below the isoelectric point the overallnet charge of the peptide will be positive.

The term “reconstituted” as used herein referring to a pharmaceuticalcomposition means an aqueous composition which has been formed by theaddition of water to a solid material comprising the activepharmaceutical ingredient. Pharmaceutical compositions forreconstitution are applied where a liquid composition with acceptableshelf-life cannot be produced. An example of a reconstitutedpharmaceutical composition is the solution which results when addingwater to a freeze dried composition. The solution is often forparenteral administration and thus water for injection is typically usedfor reconstituting the solid material.

The term “about” as used herein in relation to the concentration of apeptide in a pharmaceutical composition means plus or minus 10%. Hence,the concentration “about 5 mg/mL insulin” means a concentration of 4.5mg/mL insulin to 5.5 mg/mL insulin.

DESCRIPTION OF THE INVENTION

In one aspect the invention relates to a pharmaceutical composition forparenteral administration, which comprises an insulinotropic peptide, ameal-related insulin peptide, a pharmaceutically acceptable preservativeand optionally an isotonicity agent.

In embodiment of the invention the pH of said pharmaceutical compositionor a reconstituted solution of said pharmaceutical composition is frompH 7.0 to pH 9.0.

In embodiment of the invention the pH of said pharmaceutical compositionor a reconstituted solution of said pharmaceutical composition is frompH 7.0 to pH 8.0.

In another embodiment of the invention the composition is a solution. Inanother embodiment of the invention the pharmaceutical composition is asolid. In another embodiment of the invention the pharmaceuticalcomposition is to be reconstituted with an aqueous solution such as abuffer or water for injection.

In another embodiment of the invention the pharmaceutical composition issuitable for administration by injection or infusion. In anotherembodiment of the invention the pharmaceutical composition is suitablefor subcutaneous administration. In another embodiment of the inventionthe pharmaceutical composition is suitable for intramuscularadministration. In another embodiment of the invention thepharmaceutical composition is suitable for intravenous administration.

In a further embodiment the present invention relates to apharmaceutical composition wherein the meal-related insulin peptide hasa time action of less than 4 hours.

In another aspect the present invention relates to a pharmaceuticalcomposition wherein said insulin peptide is human insulin, an analogueof human insulin, a derivative of human insulin or a derivative of ahuman insulin analogue.

In one embodiment of the invention said insulin peptide is humaninsulin.

In a further aspect the present invention relates to a pharmaceuticalcomposition wherein said insulin peptide is a human insulin analogue. Inone embodiment of the invention said human insulin analogue isAsp^(B28)-human insulin. In another embodiment of the invention saidhuman insulin analogue is Lys^(B28), Pro^(B29)-human insulin. In anotherembodiment of the invention said human insulin analogue is Lys^(B3),Glu^(B29)-human insulin. In another embodiment of the invention saidhuman insulin analogue is des(B30) human insulin. In another embodimentof the invention said insulin peptide is a derivative of a human insulinanalogue.

In another embodiment of the invention the concentration of saidmeal-related insulin peptide in said pharmaceutical composition is inthe range from about 1.6 mg/mL to about 5.6 mg/mL, or from about 2.6mg/mL to about 4.6 mg/mL, or from about 3.2 mg/mL to about 4.0 mg/mL.

In another embodiment of the invention the concentration of saidmeal-related insulin peptide in said pharmaceutical composition is inthe range from about 1 mg/mL to about 10 mg/mL, or from about 2.5 mg/mLto about 8.75 mg/mL, or from about 3.5 mg/mL to about 8.75 mg/mL, orfrom about 5 mg/mL to about 8.75 mg/mL.

In another embodiment of the invention the pharmaceutical compositioncomprises two different insulin peptides.

In another aspect the present invention relates to a pharmaceuticalcomposition wherein said insulinotropic peptide is GLP-1(7-37) (SEQ IDNO. 1), a GLP-1(7-37) analogue, a derivative of GLP-1(7-37), or aderivative of a GLP-1(7-37) analogue. In another embodiment of theinvention said GLP-1(7-37) analogue is selected from the groupconsisting of Arg³⁴-GLP-1(7-37), Gly⁸-GLP-1(7-36)-amide,Gly⁸-GLP-1(7-37), Val⁸-GLP-1(7-36)-amide, Val⁸-GLP-1(7-37),Val⁸Asp²²-GLP-1(7-36)-amide, Val⁸Asp²²-GLP-1(7-37),Val⁸Glu²²-GLP-1(7-36)-amide, Val⁸Glu²²-GLP-1(7-37),Val⁸Lys²²-GLP-1(7-36)-amide, Val⁸Lys²²-GLP-1(7-37),Val⁸Arg²²-GLP-1(7-36)-amide, Val⁸Arg²²-GLP-1(7-37),Val⁸His²²-GLP-1(7-36)-amide, Val⁸His²²-GLP-1(7-37),Val⁸Trp¹⁹Glu²²-GLP-1(7-37), Val⁸Glu²²Val²⁵-GLP-1(7-37),Val⁸Tyr¹⁶Glu²²-GLP-1(7-37), Val⁸Trp¹⁶Glu²²-GLP-1(7-37),Val⁸Leu¹⁶Glu²²-GLP-1(7-37), Val⁸Tyr¹⁸Glu²²-GLP-1(7-37),Val⁸Glu¹²His³⁷-GLP-1(7-37), Val⁸Glu²²Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Val²⁵Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Ile³³-GLP-1(7-37),Val⁸Glu²²Val²⁵Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Val²⁵-GLP-1(7-37),analogues thereof and derivatives of any of these.

In another embodiment of the invention said insulinotropic peptide has aGlu residue in position 22. In another embodiment of the invention saidinsulinotropic peptide has a L-histidine residue in position 8. Inanother embodiment of the invention said insulinotropic peptide has aVal residue in position 8. In another embodiment of the invention saidderivative of a GLP-1(7-37) analogue is GLP-1(7-36)-amide.

In another aspect the present invention relates to a pharmaceuticalcomposition wherein said insulinotropic peptide is a derivative ofGLP-1(7-37) or a derivative of a GLP-1(7-37) analogue having a lysineresidue, such as one lysine, wherein a lipophilic substituent optionallyvia a spacer is attached to the epsilon amino group of said lysine. Inone embodiment of the invention said lipophilic substituent has from 8to 40 carbon atoms, preferably from 8 to 24, eg 12-18. In anotherembodiment of the invention said spacer is present and is selected froman amino acid, eg. beta-Ala, L-Glu, aminobutyroyl. In another embodimentof the invention said insulinotropic peptide is a dipeptidylaminopeptidase IV protected GLP-1 compound. In another embodiment of theinvention said insulinotropic peptide is a plasma stable GLP-1 compound.In another embodiment of the invention said derivative of a GLP-1(7-37)analogue is Arg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).In another embodiment of the invention said insulinotropic peptide hasfrom 27 to 43 amino acid residues, preferable from 28 to 38 amino acidresidues, even more preferable from 30 to 34 amino acid residues.

In another embodiment of the invention the concentration of saidinsulinotropic peptide in said pharmaceutical composition is from about1 mg/mL to about 25 mg/mL, from about 2 mg/mL to about 15 mg/mL, fromabout 5 mg/mL to about 12 mg/mL, or from about 8 mg/mL to about 11mg/mL. In another embodiment of the invention the concentration of saidinsulinotropic peptide in said pharmaceutical composition is from about5 mg/mL to about 7.5 mg/mL.

In another aspect the present invention relates to a pharmaceuticalcomposition wherein said insulinotropic peptide is exendin-4 (SEQ ID NO.2), an exendin-4 analogue, a derivative of exendin-4, or a derivative ofan exendin-4 analogue. In one embodiment of the invention saidinsulinotropic peptide is exendin-4 (SEQ ID NO. 2). In anotherembodiment of the invention said exendin-4 analogue is exendin-3 orZP-10 (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2, SEQ ID NO. 3).

In another embodiment of the invention said derivative of an exendin-4analogue is an acylated exendin-4 analogue or a pegylated exendin-4analogue. In another embodiment of the invention said insulinotropicpeptide is a derivative of exendin-4 or a derivative of an exendin-4analogue having a lysine residue, such as one lysine, wherein alipophilic substituent optionally via a spacer is attached to theepsilon amino group of said lysine. In another embodiment of theinvention said lipophilic substituent has from 8 to 40 carbon atoms,preferably from 8 to 24, eg 12-18. In another embodiment of theinvention said spacer is present and is selected from an amino acid, eg.beta-Ala, L-Glu, aminobutyroyl. In another embodiment of the inventionsaid insulinotropic peptide is a dipeptidyl aminopeptidase IV protectedexendin-4 compound. In another embodiment of the invention saidinsulinotropic peptide is a plasma stable exendin-4 compound. In anotherembodiment of the invention said insulinotropic peptide has from 30 to48 amino acid residues, from 33 to 45 amino acid residues, preferablefrom 35 to 43 amino acid residues, even more preferable from 37 to 41amino acid residues. In another embodiment of the invention theconcentration of said insulinotropic peptide in said pharmaceuticalcomposition is from about 5 μg/mL to about 10 mg/mL, from about 5 μg/mLto about 5 mg/mL, from about 0.1 mg/mL to about 3 mg/mL, or from about0.2 mg/mL to about 1 mg/mL.

In another aspect the present invention relates to a pharmaceuticalcomposition wherein the isoelectric point of said insulinotropic peptideis from 3.0 to 7.0, from 4.0 to 6.0, preferable from 4.2 to 5.5, evenmore preferable from 4.3 to 5.2.

In one aspect the present invention relates to a pharmaceuticalcomposition which further comprises zinc. In one embodiment of theinvention the molar ratio of zinc to insulin peptide is from ⅙ to ½mole/mole, preferable from 3/12 to 5/12 mole/mole.

In another aspect the present invention relates to a pharmaceuticalcomposition, wherein said meal-related insulin peptide is human insulinand said insulinotropic peptide is Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37). In one embodimentof the invention the concentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom from about 1 mg/mL to about 25 mg/mL and the concentration of humaninsulin is in the range from about 3.2 mg/mL to about 4.0 mg/mL. Inanother embodiment of the invention the concentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom from about 5 mg/mL to about 15 mg/mL and the concentration of humaninsulin is in the range from about 3.2 mg/mL to about 4.0 mg/mL.

In another aspect the present invention relates to a pharmaceuticalcomposition, wherein said insulin peptide is Asp^(B28)-human insulin andsaid insulinotropic peptide is Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37). In one embodimentof the invention the concentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom from about 1 mg/mL to about 25 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.2 mg/mL to about4.0 mg/mL. In another embodiment of the invention the concentration ofArg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in therange from from about 5 mg/mL to about 15 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.2 mg/mL to about4.0 mg/mL. In another embodiment of the invention the concentration ofArg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in therange from about 1 mg/mL to about 25 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.4 mg/mL to about3.8 mg/mL. In another embodiment of the invention the concentration ofArg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in therange from about 5 mg/mL to about 15 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.4 mg/mL to about3.8 mg/mL.

In another aspect the present invention relates to a pharmaceuticalcomposition, wherein said insulin peptide is Lys^(B3), Glu^(B29)-humaninsulin and said insulinotropic peptide is ZP-10(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2, SEQ ID NO. 3). In oneembodiment of the invention the concentration of Lys^(B3),Glu^(B29)-human insulin is in the range from about 3.2 mg/mL to about4.0 mg/mL. In another embodiment of the invention the concentration ofZP-10 is in the range from about 0.1 mg/mL to about 3 mg/mL.

In another embodiment of the present invention the preservative isphenol, m-cresol or a mixture thereof.

In another aspect of the present invention the pharmaceuticalcomposition comprises a buffer. In one embodiment of the invention saidbuffer is phosphate, TRIS, HEPES, glycine, N-glycylglycine, citrate ormixtures thereof.

In another aspect of the present invention the pharmaceuticalcomposition comprises an isotonicity agent. In one embodiment theisotonicity agent is not a salt. In another embodiment the isotonicityagent is selected from mannitol, sorbitol, glycerol, propylene glycol ora mixture thereof.

In another aspect the present invention relates to a solublepharmaceutical composition for parenteral administration, whichcomprises an insulinotropic peptide, a meal-related insulin peptide, apharmaceutically acceptable preservative, a stabiliser and optionally anisotonicity agent. In one embodiment of the invention said stabiliser isselected from the group consisting of L-histidine, imidazole andL-arginine. In another embodiment of the invention said stabiliser is apolyethylene glycol.

In another aspect the present invention relates to a solublepharmaceutical composition which comprises an insulinotropic peptide, ameal-related insulin peptide, a pharmaceutically acceptablepreservative, a surfactant and optionally an isotonicity agent.

In one embodiment the surfactant is a poloxamer.

In another embodiment the surfactant is a poloxamer 188.

In another embodiment the surfactant is selected from the groupconsisting of poloxamer 407, poloxamer 124, poloxamer 181, poloxamer182, poloxamer 237, poloxamer 331 and poloxamer 338.

In another embodiment the surfactant is a polysorbate 20 (Tween-20).

In another embodiment the concentration of said surfactant is from about5 mg/L to about 3000 mg/L.

In another embodiment the concentration of said surfactant is from about10 mg/L to about 500 mg/L.

In another embodiment the concentration of said surfactant is from about20 mg/L to about 300 mg/L.

In another embodiment the concentration of said surfactant is from about50 mg/L to about 200 mg/L.

In another embodiment the pharmaceutical composition comprises twodifferent surfactants.

In another embodiment the pharmaceutical composition comprises poloxamer188 and polysorbate 20 (Tween-20).

In another embodiment of the invention the surfactant is selected from adetergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylatedmonoglycerides, sorbitan fatty acid esters, poloxamers, such aspoloxamer 188 and poloxamer 407, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene derivatives such as alkylated and alkoxylatedderivatives (tweens, e.g. Tween-20, or Tween-80), monoglycerides orethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, glycerol, cholic acid or derivatives thereof,lecithins, alcohols and phospholipids, glycerophospholipids (lecithins,kephalins, phosphatidyl serine), glyceroglycolipids (galactopyransoide),sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides,gangliosides), DSS (docusate sodium, CAS registry no [577-11-7]),docusate calcium, CAS registry no [128-49-4]), docusate potassium, CASregistry no [7491-09-0]), SDS (sodium dodecyl sulfate or sodium laurylsulfate), dipalmitoyl phosphatidic acid, sodium caprylate, bile acidsand salts thereof and glycine or taurine conjugates, ursodeoxycholicacid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of Iysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the postively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine,zwitterionic surfactants (e.g.N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egglysolecithin), cationic surfactants (quarternary ammonium bases) (e.g.cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionicsurfactants, polyethyleneoxide/polypropyleneoxide block copolymers(Pluronics/Tetronics, Triton X-100, Dodecyl β-D-glucopyranoside) orpolymeric surfactants (Tween-40, Tween-80, Brij-35), fusidic acidderivatives—(e.g. sodium taurodihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, or thesurfactant may be selected from the group of imidazoline derivatives, ormixtures thereof. Each one of these specific surfactants constitutes analternative embodiment of the invention. In a further embodiment of theinvention the surfactant is a polysorbate, such as polysorbate-20. In afurther embodiment of the invention the pharmaceutical compositioncomprises a surfactant in a concentration from about 1 ppm to about 500ppm, preferably from about 10 ppm to about 120 ppm.

In another aspect the present invention relates to a method fortreatment of hyperglycemia comprising parenteral administration of aneffective amount of a pharmaceutical composition, which comprises aninsulinotropic peptide, a meal-related insulin peptide, apharmaceutically acceptable preservative, and optionally an isotonicityagent.

In another aspect the present invention relates to a method fortreatment of binge eating or bulimia comprising parenteraladministration of an effective amount of a pharmaceutical composition,which comprises an insulinotropic peptide, a meal-related insulinpeptide, a pharmaceutically acceptable preservative, and optionally anisotonicity agent.

In another aspect the present invention relates to a method fortreatment or prevention of type 2 diabetes, impaired glucose tolerance,type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia,cognitive disorders, atheroschlerosis, myocardial infarction, coronaryheart disease and other cardiovascular disorders, stroke, inflammatorybowel syndrome, dyspepsia and gastric ulcers comprising parenteraladministration of an effective amount of a pharmaceutical composition,which comprises an insulinotropic peptide, a meal-related insulinpeptide, a pharmaceutically acceptable preservative, and optionally anisotonicity agent.

In another aspect the present invention relates to a method for delayingor preventing disease progression in type 2 diabetes comprisingparenteral administration of an effective amount of a pharmaceuticalcomposition, which comprises an insulinotropic peptide, a meal-relatedinsulin peptide, a pharmaceutically acceptable preservative, andoptionally an isotonicity agent.

In another aspect the present invention relates to a method fordecreasing food intake, decreasing β-cell apoptosis, increasing β-cellfunction and β-cell mass, and/or for restoring glucose sensitivity toβ-cells comprising parenteral administration of an effective amount of apharmaceutical composition, which comprises an insulinotropic peptide, ameal-related insulin peptide, a pharmaceutically acceptablepreservative, and optionally an isotonicity agent.

When the pharmaceutical compositions according to the present inventionare administered by injection, e.g. via a pen or a syringe, it istypically administered 3 times per day, preferably before meals. It ispreferred that each administration comprises less than about 500 μL, orless than about 200 μL since larger injection volumes are unpleasant forthe patient. When the pharmaceutical compositions according to thepresent invention are administered by a pump, it is typicallyadministrered continuously or discontinuously via at least 10administrations or more per day. In one embodiment of the invention themethod of treatment comprises administration of an effective amount ofthe pharmaceutical composition which is from about 30 μL/day to about600 μL/day, such as from about 60 μL/day to about 360 μL/day. In anotherembodiment of the invention the method comprises a pharmaceuticalcomposition for administration by subcutaneous injection. In anotherembodiment of the invention the method comprises a pharmaceuticalcomposition for administration by a pump. In another embodiment of theinvention the method comprises administration by a pump which delivers adiscontinuous amount of said pharmaceutical composition. In anotherembodiment of the invention the method comprises administration by apump which delivers a discontinuous amount of said pharmaceuticalcomposition wherein said discontinuous administration of saidpharmaceutical composition is by a pulse dosing for a period of timewhich is less than the period between pulses.

In another aspect the present invention relates to the use of aninsulinotropic peptide and a meal-related insulin peptide for themanufacture of a pharmaceutical composition for parenteraladministration, which composition comprises an insulinotropic peptide, ameal-related insulin peptide, a pharmaceutically acceptablepreservative, and optionally an isotonicity agent. In one embodiment ofthe invention the use comprises a pharmaceutical composition foradministration by subcutaneous injection. In another embodiment of theinvention the use comprises a pharmaceutical composition foradministration by a pump. In another embodiment of the invention the usecomprises administration by a pump which delivers a discontinuous amountof said pharmaceutical composition. In another embodiment of theinvention the use comprises administration by a pump which delivers adiscontinuous amount of said pharmaceutical composition wherein saiddiscontinuous administration of said pharmaceutical composition is by apulse dosing for a period of time which is less than the period betweenpulses.

In another aspect the present invention relates to the use of an insulinpeptide and an insulinotropic peptide for the manufacture of apharmaceutical composition for the treatment of hyperglycemia byparenteral administration, which composition comprises an insulinpeptide, an insulinotropic peptide, a pharmaceutically acceptablebuffer, a pharmaceutically acceptable preservative, a surfactant andoptionally an isotonicity agent.

EXAMPLES Example 1 In Vivo Pig Data of Mixtures

Two mixtures of insulin aspart (Asp^(B28)-human insulin) and Liraglutide(Arg³⁴Lys²⁶N^(ε)(γ-Glu(N^(α)-hexadecanoyl))GLP-1(7-37)), with insulinaspart labelled in Tyr A14 with ¹²⁵I, were prepared for subcutaneousadministration to pigs. The study design was simultaneous measuring ofinsulin aspart disappearance from the injection site by external gammacounting, and monitoring of Liraglutide plasma concentration time coursefor up to 72 hours.

Aspart Formulation (Reference)

600 μM Aspart (with tracer), 300 μM Zn(Ac)₂, pH 7.4, 30 mM phenol, 1.6%glycerol.

Mix Aspart:Liraglutide Formulation, 1:1

600 μM Aspart (with tracer), 600 μM Liraglutide, 300 μM Zn(Ac)₂, pH 7.4,30 mM phenol, 1.6% glycerol.

Mix Aspart:Liraglutide Formulation, 1:5

600 μM Aspart (with tracer), 3000 μM Liraglutide, 300 μM Zn(Ac)₂, pH7.4, 30 mM phenol, 1.6% glycerol.

Six normal and healthy pigs (Dansk Landrace, LDY) having a body weightof 80 kg to 100 kg were included in the study. The pigs were eachadministered 100 μL insulin aspart formulation (reference) at one sideof the neck and 100 μL mix aspart/liraglutide formulation at theopposite side. Disappearance courses of radiolabelled insulin aspartwere measured at both injection sites in 6 pigs for the insulin aspartreference, and in 3 pigs for each mix 1:1 and 1:5. Simultaneously bloodsamples were collected over a period of 72 hours to monitor the plasmaconcentration time course of Liraglutide.

Average Disappearance Curves with Standard Deviations

The average disappearance curves are shown in FIG. 1. The disappearanceversus time of insulin aspart from the subcutaneous tissue followingadministration of the 1:5 mixture was only slightly slower than for boththe reference and the 1:1 mixture. The mean (SD) time point for 75%, 50%and 25% of the remaining radioactivity following injection, werecalculated, please refer to table 1 below. TABLE 1 Summary ofdisappearance study. Insulin Aspart reference Mix 1:1 Mix 1:5 (n = 6) (n= 3) (n = 3) T75% (h) 0.75 ± 0.05 0.79 ± 0.07 0.83 ± 0.03 T50% (h) 1.60± 0.11 1.70 ± 0.13 1.78 ± 0.05 T25% (h) 2.79 ± 0.26 2.96 ± 0.23 3.14 ±0.18Pharmacokinetics of Liraglutide

Pharmacokinetics were calculated on plasma concentration time data.Based on AUC it was concluded that absorption of liraglutide wasindependent of the mixture applied, since liraglutide from the 1:5formulation give rise to a 5-fold increase in AUC, thereby confirmingunchanged relative bioavailability of Liraglutide.

Based on above PK parameters, a simulation was performed illustratingco-administration (with Aspart) of 0.2 nmol/kg Liraglutide at mealtimes(breakfast, lunch, and dinner). App. 0.2 nmol/kg of Liraglutide at mealtime are providing a satisfying Liraglutide plasma level indicating that1:1 is the most likely Aspart:Liraglutide ratio.

FIGS. 2 and 3 show the plasma levels of liraglutide after subcutaneousinjections of the mixtures described above. TABLE 2 Liraglutide PKparameters n = 3 t_(max) C_(max) t½ AUC Extr (hr) (pmol/L) (hr) (hr *pmol/L) (%) 2) 1:1 Mean 12.0 2180 26.2 123488 18 SD 10.4 209 5.7 34562 6Harmonic Mean 8.0 2166 25.5 117918 17 3) 1:5 Mean 8.0 21187 19.2 6654359 SD 4.0 8751 2.8 59264 3 Harmonic Mean 6.5 19230 18.9 661980 8

Example 2

Two pharmaceutical compositions were prepared according to thedescription of the present invention. Both pharmaceutical compositionscomprised the meal related insulin Asp^(B28)-human insulin (aspart) andthe insulinotropic peptide Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) (liraglutide). Inboth pharmaceutical compositions aspart concentration is 0.6 mM andliraglutide concentration is 1.2 mM. Furthermore both compositionscontained 10 mM NaCl, 8 mM phosphate buffer, 14 mg/ml propylene glycol,40 mM phenol. Turbidimetric measurements of compositions withoutpoloxamer 188 (x) and with poloxamer 188 (⋄) (500 ppm) are shown for 8and 7 days, respectively (FIG. 4).

Example 3

Two pharmaceutical compositions were prepared according to thedescription of the present invention. Both pharmaceutical compositionscomprised the meal related insulin Asp^(B28)-human insulin (aspart) andthe insulinotropic peptide Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) (liraglutide). Inboth pharmaceutical compositions aspart concentration is 0.6 mM andliraglutide concentration is 1.2 mM. Furthermore both compositionscontained 0.35 mM5-[6-(5-Cyano-1H-[1,2,3]triazol-4-yl)naphthalen-2-yloxy]pentanoic acid,10 mM bicine buffer, 14 mg/ml propylene glycol, 40 mM phenol.Turbidimetric measurements of pharmaceutical compositions withoutpoloxamer 188 (x) and with poloxamer 188 (⋄) (50 ppm) were performed for13 days (FIG. 5).

Example 4

The following pharmaceutical compositions are prepared:

-   F1. 1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3    Zn/hex, aspart 0.6 mM, 8 mM bicine, 50 ppm poloxamer 188, pH 7.7.-   F2. 1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3    Zn/hex, aspart 0.6 mM, 8 mM bicine, pH 7.7.

Physical stability of the pharmaceutical compositions is evaluated bymeans of an accelerated stressed test. The stressed test is performed asa rotation test. 50 μL air is added to 5 cartridges (glass vials) ofeach formulation. The cartridges are rotated with a frequency of 30rotations per minute for 4 hours daily. The test is stopped after 22days of rotation. The inspection of the cartridges is followed daily oras required. The turbidity of the formulation is characterized bynephelometric measurement of the turbidity on a HACH Turbidimeter2100AN. The turbidity measurement of a liquid is specified in“Nephelometric Turbidity Unit” (NTU). Physical instability of theprotein is characterised by high turbidity measurements.

The experiments show that the NTU measurements increase much morerapidly in the F2 composition as compared to the NTU trace of the F1composition.

Example 5

Thioflavin T (ThT) Fibrillation Assay: Principle and Examples

Low physical stability of a peptide may lead to amyloid fibrilformation, which is observed as well-ordered, thread-like macromolecularstructures in the sample eventually resulting in gel formation. This hastraditionally been measured by visual inspection of the sample. However,that kind of measurement is very subjective and depending on theobserver. Therefore, the application of a small molecule indicator probeis much more advantageous. Thioflavin T (ThT) is such a probe and has adistinct fluorescence signature when binding to fibrils [Naiki et al.(1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309,274-284].

The time course for fibril formation can be described by a sigmoidalcurve with the following expression [Nielsen et al. (2001) Biochemistry40, 6036-6046]: $\begin{matrix}{F = {f_{i} + {m_{i}t} + \frac{f_{f} + {m_{f}t}}{1 + {\mathbb{e}}^{- {\lbrack{{({t - t_{0}})}/\tau}\rbrack}}}}} & {{Eq}.\quad(1)}\end{matrix}$

Here, F is the ThT fluorescence at the time t. The constant t₀ is thetime needed to reach 50% of maximum fluorescence. The two importantparameters describing fibril formation are the lag-time calculated byt₀−2τ and the apparent rate constant k_(app)=1/τ.

Formation of a partially folded intermediate of the peptide is suggestedas a general initiating mechanism for fibrillation. Few of thoseintermediates nucleate to form a template onto which furtherintermediates may assembly and the fibrillation proceeds. The lag-timecorresponds to the interval in which the critical mass of nucleus isbuilt up and the apparent rate constant is the rate with which thefibril itself is formed.

Sample Preparation

Samples were prepared freshly before each assay. Each sample compositionis described in the legends. The pH of the sample was adjusted to thedesired value using appropriate amounts of concentrated NaOH and HClO₄.Thioflavin T was added to the samples from a stock solution in H₂O to afinal concentration of 1 μM.

Sample aliquots of 200 μl were placed in a 96 well microtiter plate(Packard Opti-Plate™-96, white polystyrene). Usually, eight replica ofeach sample (corresponding to one test condition) were placed in onecolumn of wells. The plate was sealed with Scotch Pad (Qiagen).

Incubation and Fluorescence Measurement

Incubation at given temperature, shaking and measurement of the ThTfluorescence emission were done in a Fluoroskan Ascent FL fluorescenceplatereader (Thermo Labsystems). The temperature was adjusted to 37° C.The orbital shaking was adjusted to 960 rpm with an amplitude of 1 mm inall the presented data. Fluorescence measurement was done usingexcitation through a 444 nm filter and measurement of emission through a485 nm filter.

Each run was initiated by incubating the plate at the assay temperaturefor 10 min. The plate was measured every 20 minutes for typically 45hours. Between each measurement, the plate was shaken and heated asdescribed.

Data Handling

The measurement points were saved in Microsoft Excel format for furtherprocessing and curve drawing and fitting was performed using GraphPadPrism. The background emission from ThT in the absence of fibrils wasnegligible. The data points are typically a mean of eight samples andshown with standard deviation error bars. Only data obtained in the sameexperiment (i.e. samples on the same plate) are presented in the samegraph ensuring a relative measure of fibrillation between experiments.

The data set may be fitted to Eq. (1). However, since full sigmodialcurves in this case are not always achieved during the measurement time,the degree of fibrillation is expressed as ThT fluorescence tabulated asthe mean of the eight samples and shown with the standard deviation atvarious time points.

Example 6

A formulation of insulin aspart and liraglutide in a 1:2 mix ratio inwater adjusted to pH 7.7 is highly physical unstable as seen in FIG. 6.The ThT fluorescence signal increases instantaneously and reaches aplateau before 10 hours of assay time. However, the addition ofPoloxamer 188 stabilises this formulation. Both at 50 ppm and 200 ppmPoloxamer 188, the insulin aspart-liraglutide mix formulation does notexhibit any increase in ThT fluorescence above background level, hencethese samples are fully physical stable and do not fibrillate.

Example 7

Also Polysorbate 20 is capable of stabilising an insulinaspart-liraglutide 1:2 mix formulated in water, see FIG. 7. The presenceof 200 ppm Polysorbate 20 fully suppresses fibrillation.

Example 8

When formulating insulin aspart and liraglutide in a 1:2 mix ratio insodium phosphate buffer, the sample is highly physically unstable, seeFIG. 8. The presence of 50 ppm or 200 ppm Poloxamer 188 prolongs the lagtime before on-set of fibrillation: from the instantaneous fibrillationin the absence of Poloxamer 188, it is longer than 15 hours in thepresence of either 50 ppm or 200 ppm Poloxamer 188. The sample with thehighest Poloxamer 188 concentration exhibits the lowest ThT signal after40 hours of assay time.

Example 9

Polysorbate 20 does also stabilise insulin aspart-liraglutide 1:2 mixesformulated in sodium phosphate buffer, see FIG. 9. The presence of 100ppm Polysorbate 20 increases the lag time to longer than 5 hourscompared to the sample without Polysorbate 20. The presence of 200 ppmPolysorbate 20 fully suppresses fibrillation of the sample.

Example 10

Formulations where insulin aspart and liraglutide are mixed in a 1:5ratio are also highly physical unstable. These formulations arestabilised by the presence of 100 ppm Poloxamer 188 or 200 ppmPolysorbate 20. This is observed as a prolongation of the lag timebefore on-set of fibrillation in the presence of either Poloxamer 188 orPolysorbate 20 compared with a similar formulation without eitherPoloxamer 188 or Polysorbate 20.

1. A soluble pharmaceutical composition for parenteral administration,which comprises an insulinotropic peptide, a meal-related insulinpeptide, a pharmaceutically acceptable preservative and optionally anisotonicity agent.
 2. The pharmaceutical composition according to claim1, wherein the pH of said pharmaceutical composition or a reconstitutedsolution of said pharmaceutical composition is from about pH 7.0 toabout pH 9.0.
 3. The pharmaceutical composition according to claim 1,wherein the pH of said pharmaceutical composition or a reconstitutedsolution of said pharmaceutical composition is from about pH 7.0 toabout pH 8.0.
 4. The pharmaceutical composition according to claim 1,wherein the composition is a solution.
 5. The pharmaceutical compositionaccording to claim 1, wherein the composition is a solid.
 6. Thepharmaceutical composition according to claim 5, which is to bereconstituted with an aqueous solution such as a buffer or water forinjection.
 7. The pharmaceutical composition according to claim 1, whichis suitable for administration by injection or infusion.
 8. Thepharmaceutical composition according to claim 1, wherein saidmeal-related insulin peptide has a time action of less than 4 hours. 9.The pharmaceutical composition according to claim 1, wherein saidmeal-related insulin peptide is human insulin, an analogue of humaninsulin, a derivative of human insulin or a derivative of a humaninsulin analogue.
 10. The pharmaceutical composition according to claim9, wherein said meal-related insulin peptide is human insulin.
 11. Thepharmaceutical composition according to claim 9, wherein saidmeal-related insulin peptide is a human insulin analogue.
 12. Thepharmaceutical composition according to claim 11, wherein saidmeal-related human insulin analogue is Asp^(B28)-human insulin.
 13. Thepharmaceutical composition according to claim 11, wherein saidmeal-related human insulin analogue is Lys^(B28), Pro^(B29)-humaninsulin.
 14. The pharmaceutical composition according to claim 11,wherein said meal-related human insulin analogue is Lys^(B3),Glu^(B29)-human insulin.
 15. The pharmaceutical composition according toclaim 11, wherein said meal-related human insulin analogue is des(B30)human insulin.
 16. The pharmaceutical composition according to claim 9,wherein said meal-related insulin peptide is a derivative of a humaninsulin analogue.
 17. The pharmaceutical composition according to claim1, wherein the concentration of said meal-related insulin peptide is inthe range from about 1.6 mg/mL to about 5.6 mg/mL.
 18. Thepharmaceutical composition according claim 1, wherein the concentrationof said meal-related insulin peptide is in the range from about 1 mg/mLto about 10 mg/mL.
 19. The pharmaceutical composition according to claim1, wherein said composition comprises two different insulin peptides.20. The pharmaceutical composition according to claim 1, wherein saidinsulinotropic peptide is GLP-1(7-37) (SEQ ID NO. 1), a GLP-1(7-37)analogue, a derivative of GLP-1(7-37), or a derivative of a GLP-1(7-37)analogue.
 21. The pharmaceutical composition according to claim 20,wherein said GLP-1(7-37) analogue is selected from the group consistingof Arg³⁴-GLP-1(7-37), Gly⁸-GLP-1(7-36)-amide, Gly⁸-GLP-1(7-37),Val⁸-GLP-1(7-36)-amide, Val⁸-GLP-1(7-37), Val⁸Asp²²-GLP-1(7-36)-amide,Val⁸Asp²²-GLP-1(7-37), Val⁸Glu²²-GLP-1(7-36)-amide,Val⁸Glu²²-GLP-1(7-37), Val⁸Lys²²-GLP-1(7-36)-amide,Val⁸Lys²²-GLP-1(7-37), Val⁸Arg²²-GLP-1(7-36)-amide,Val⁸Arg²²-GLP-1(7-37), Val⁸His²²-GLP-1(7-36)-amide,Val⁸His²²-GLP-1(7-37), Val⁸Trp¹⁹Glu²²-GLP-1(7-37),Val⁸Glu²²Val²⁵-GLP-1(7-37), Val⁸Tyr¹⁶Glu²²-GLP-1(7-37),Val⁸Trp¹⁶Glu²²-GLP-1(7-37), Val⁸Leu¹⁶Glu²²-GLP-1(7-37),Val⁸Tyr¹⁸Glu²²-GLP-1(7-37), Val⁸Glu²²His³⁷-GLP-1(7-37),Val⁸Glu²²Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Val²⁵Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Ile³³-GLP-1(7-37), Val⁸Glu²²Val²⁵Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Val²⁵-GLP-1(7-37), analogues thereof and derivatives ofany of these.
 22. The pharmaceutical composition according to claim 20,wherein said derivative of a GLP-1(7-37) analogue is GLP-1(7-36)-amide.23. The pharmaceutical composition according to claim 1, wherein saidinsulinotropic peptide is a derivative of GLP-1(7-37) or a derivative ofa GLP-1(7-37) analogue having a lysine residue and wherein a lipophilicsubstituent optionally via a spacer is attached to the epsilon aminogroup of said lysine.
 24. The pharmaceutical composition according toclaim 23, wherein said lipophilic substituent has from 8 to 40 carbonatoms.
 25. The pharmaceutical composition according to claim 23, whereinsaid spacer is present and is selected from an amino acid.
 26. Thepharmaceutical composition according to claim 1, wherein saidinsulinotropic peptide is a dipeptidyl aminopeptidase IV protected GLP-1compound.
 27. The pharmaceutical composition according to claim 1,wherein said insulinotropic peptide is a plasma stable GLP-1 compound.28. The pharmaceutical composition according to any one of claim 20,wherein said derivative of a GLP-1(7-37) analogue is Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).
 29. Thepharmaceutical composition according to claim 20, wherein saidinsulinotropic peptide has from 27 to 43 amino acid residues.
 30. Thepharmaceutical composition according to claim 20, wherein theconcentration of said insulinotropic peptide is from about 1 mg/mL toabout 25 mg/mL.
 31. The pharmaceutical composition according to claim 1,wherein said insulinotropic peptide is exendin-4 (SEQ ID NO 2), anexendin-4 analogue, a derivative of exendin-4, or a derivative of anexendin-4 analogue.
 32. The pharmaceutical composition according toclaim 31, wherein said insulinotropic peptide is exendin-4 (SEQ ID NO.2).
 33. The pharmaceutical composition according to claim 31, whereinsaid exendin-4 analogue is exendin-3 or ZP-10(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2, SEQ ID. NO. 3). 34.The pharmaceutical composition according to claim 31, wherein saidderivative of an exendin-4 analogue is an acylated exendin-4 analogue ora pegylated exendin-4 analogue.
 35. The pharmaceutical compositionaccording to claim 31, wherein said insulinotropic peptide is aderivative of exendin-4 or a derivative of an exendin-4 analogue havinga lysine residue, and wherein a lipophilic substituent optionally via aspacer is attached to the epsilon amino group of said lysine.
 36. Thepharmaceutical composition according to claim 35, wherein saidlipophilic substituent has from 8 to 40 carbon atoms.
 37. Thepharmaceutical composition according to claim 35, wherein said spacer ispresent and is selected from an amino acid.
 38. The pharmaceuticalcomposition according to claim 1, wherein said insulinotropic peptide isa dipeptidyl aminopeptidase IV protected exendin-4 compound.
 39. Thepharmaceutical composition according to claim 1, wherein saidinsulinotropic peptide is a plasma stable exendin-4 compound.
 40. Thepharmaceutical composition according to claim 31, wherein saidinsulinotropic peptide has from 30 to 48 amino acid residues.
 41. Thepharmaceutical composition according to claim 20, wherein theconcentration of said insulinotropic peptide is from about 5 μg/mL toabout 10 mg/mL.
 42. The pharmaceutical composition according to claim 1,wherein the isoelectric point of said insulinotropic peptide is from 3.0to 7.0.
 43. The pharmaceutical composition according to claim 1, saidcomposition further comprising zinc.
 44. The pharmaceutical compositionaccording to claim 43, wherein the molar ratio of zinc to insulinpeptide is from ⅙ to ½ mole/mole.
 45. The pharmaceutical compositionaccording to claim 1, wherein said meal-related insulin peptide is humaninsulin and said insulinotropic peptide is Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).
 46. Thepharmaceutical composition according to claim 45, wherein theconcentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom from about 5 mg/mL to about 15 mg/mL and the concentration of humaninsulin is in the range from about 3.2 mg/mL to about 4.0 mg/mL.
 47. Thepharmaceutical composition according to claim 1, wherein said insulinpeptide is Asp^(B28)-human insulin and said insulinotropic peptide isArg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).
 48. Thepharmaceutical composition according to claim 47, wherein theconcentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom about 5 mg/mL to about 15 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.2 mg/mL to about4.0 mg/mL.
 49. The pharmaceutical composition according to claim 47,wherein the concentration of Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) is in the rangefrom about 5 mg/mL to about 15 mg/mL and the concentration ofAsp^(B28)-human insulin is in the range from about 3.4 mg/mL to about3.8 mg/mL.
 50. The pharmaceutical composition according to claim 1,wherein said insulin peptide is Lys^(B3), Glu^(B29)-human insulin andsaid insulinotropic peptide is ZP-10(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2, SEQ ID. NO. 3). 51.The pharmaceutical composition according to claim 50, wherein theconcentration of Lys^(B3), Glu^(B29)-human insulin is in the range fromabout 3.2 mg/mL to about 4.0 mg/mL.
 52. The pharmaceutical compositionaccording to claim 50, wherein the concentration of ZP-10 is in therange from about 0.1 mg/mL to about 3 mg/mL.
 53. The pharmaceuticalcomposition according to claim 1, wherein said preservative is phenol,m-cresol or a mixture thereof.
 54. The pharmaceutical compositionaccording to claim 1, wherein said pharmaceutical composition comprisesa buffer.
 55. The pharmaceutical composition according to claim 54,wherein said buffer is phosphate, TRIS, HEPES, glycine, N-glycylglycine,citrate or a mixture thereof.
 56. The pharmaceutical compositionaccording to claim 1, wherein said pharmaceutical composition comprisesan isotonicity agent.
 57. The pharmaceutical composition according toclaim 56, wherein said isotonicity agent is not a salt.
 58. Thepharmaceutical composition according to claim 57, wherein saidisotonicity agent is selected from mannitol, sorbitol, glycerol,propylene glycol or a mixture thereof.
 59. The pharmaceuticalcomposition according to claim 1, wherein said composition furthercomprises a stabiliser.
 60. The pharmaceutical composition according toclaim 59, wherein said stabiliser is selected from the group consistingof L-histidine, imidazole and L-arginine.
 61. The pharmaceuticalcomposition according to claim 59, wherein said stabiliser is apolyethylene glycol.
 62. The pharmaceutical composition according toclaim 1, wherein said composition further comprises a surfactant. 63.The pharmaceutical composition according to claim 62, wherein saidsurfactant is a poloxamer.
 64. The pharmaceutical composition accordingto claim 63, wherein said surfactant is a poloxamer
 188. 65. Thepharmaceutical composition according to claim 63, wherein saidsurfactant is selected from the group consisting of poloxamer 407,poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 237, poloxamer331 and poloxamer
 338. 66. The pharmaceutical composition according toclaim 62, wherein said surfactant is a polysorbate 20 (Tween-20). 67.The pharmaceutical composition according to claim 62, wherein theconcentration of said surfactant is from about 5 mg/L to about 3000mg/L.
 68. The pharmaceutical composition according to claim 62, whereinthe concentration of said surfactant is from about 10 mg/L to about 500mg/L.
 69. The pharmaceutical composition according to claim 62, whereinthe concentration of said surfactant is from about 20 mg/L to about 300mg/L.
 70. The pharmaceutical composition according to claim 62, whereinthe concentration of said surfactant is from about 50 mg/L to about 200mg/L.
 71. The pharmaceutical composition according to claim 62, whereinsaid composition comprises two different surfactants.
 72. Thepharmaceutical composition according to claim 71, comprising poloxamer188 and polysorbate 20 (Tween-20).
 73. A method for treatment ofhyperglycemia comprising parenteral administration of an effectiveamount of the pharmaceutical composition according to claim
 1. 74. Themethod according to claim 73, wherein said effective amount of thepharmaceutical composition is from about 30 μL/day to about 600 μL/day.75. The method according to claim 73, wherein administration is bysubcutaneous injection.
 76. The method according to claim 73, whereinadministration is by a pump.
 77. The method according to claim 73,wherein administration is by a pump which delivers a discontinuousamount of said pharmaceutical composition.
 78. The method according toclaim 77, wherein said discontinuous administration of saidpharmaceutical composition is by a pulse dosing for a period of timewhich is less than the period of time between pulses.
 79. A method fortreatment of binge eating or bulimia, said method comprising parenteraladministration of an effective amount of the pharmaceutical compositionaccording to claim 1